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Distinct steady–state nuclear receptor coregulator complexesexistinvivo

Neil J McKenna1,Zafar Nawaz1,Sophia Y Tsai1,Ming-Jer Tsai1,Bert W O’Malley1,*
1Department of Cell Biology, Baylor College of Medicine, One Baylor Plaza, Houston, TX 77030
*

To whom reprint requests should be addressed. e-mail:berto@bcm.tmc.edu.

Contributed by Bert W. O’Malley

Accepted 1998 Aug 7.

Copyright © 1998, The National Academy of Sciences
PMCID: PMC21703  PMID:9751728

Abstract

Transcriptional regulation by members of the nuclear hormonereceptor superfamily is a modular process requiring the mediation ofdistinct subclasses of coregulators. These subclasses include membersof the steroid receptor coactivator-1 (SRC-1) coactivator family,p300/CBP and their associated proteins, such as p300/CBP-associatedfactor, human homologs of SWI/SNF proteins such as BRG-1, and theless well-characterized E3 ubiquitin-protein ligases E6 papillomavirusprotein-associated protein and receptor-potentiating factor-1. Becausefunctional studies indicate that these coregulators may form higherorder complexes, we analyzed steady–state complexes of differentcoregulator subclassesin vivo. T47D and HeLa celllysates were subjected to biochemical fractionation and screened byimmunoblotting using coregulator-specific antibodies. We show thatdifferent subclasses of nuclear receptor coregulators exhibit distinctfractionation profiles. Furthermore, evidence is provided that SRC-1family members may existin vivo in heteromultimericforms with each other. In addition, we demonstrate that liganded PR ispresent in stable complexes containing SRC-1 and transcriptionintermediary factor 2 (TIF2)in vivo. Our resultssuggest that the assembly of large, modular transcriptional complexesby recruitment of distinct subclasses of preformed coregulatorsubcomplexes may be involved in transcriptional regulation by activatednuclear receptors.

Members of the nuclear receptor family of ligand-inducibletranscription factors activate transcription in response to theirligands via enhancer elements located in the promoters of target genes(1). Recently it has become clear that transactivation by thesereceptors is a modular process, requiring interaction with an array ofcofactors capable of (i) modifying the chromatin structureof hormone-regulated promoters by intrinsic histone acetyltransferase(HAT) activities, (ii) mediating interactions between thereceptors and other transcription factors, and (iii)directing assembly and stabilization of the transcriptionalpreinitiation complex. Several structurally distinct subclasses ofnuclear receptor coregulators have been identified, including: membersof the steroid receptor coactivator-1 (SRC-1) family, the cointegratorsp300 and CBP and their associated proteins; mammalian homologs of yeastSWI/SNF proteins; and the less well characterized E3ubiquitin-protein ligase coactivators.

Our laboratory initially cloned SRC-1 as a factor required fortransactivation by nuclear receptors (2), and SRC-1 has been termedvariously as p160/NCoA-1 (3), and ERAP-160 (4). The subsequentidentification of two more members of the SRC-1 family, namelytranscription intermediary factor-2 [TIF2/GRIP-1/SRC-2] (5–7),and p/CIP (8) [ACTR (9)/RAC-3 (10)/AIB-1 (11)/TRAM-1(12)/SRC-3] established the existence of a class of structurally andfunctionally related nuclear receptor coactivators. Sequence alignmentof the members of the SRC-1 family highlights the shared domainstructure throughout and predicts common modes of action by theindividual members. SRC-1 family members have C-terminal domains thatcontain HAT activity, suggesting that they modify chromatin (9,13).The presence in their extreme N termini of a postulated multimerizationmotif, the Per-Arnt Sim/basic helix–loop–helix homology domain(14), implies that molecular interactions between SRC-1 family membersand other Per-Arnt-Sim/basic helix–loop–helix homology domainproteins might be important for their functionin vivo.

A class of coregulators structurally distinct from the SRC-1 family,the cointegrators, is defined by the functionally related proteins p300and CBP. These proteins exhibit broad functional specificity inaddition to extensive amino acid sequence identity (15,16) and areproposed to function by adapting signaling pathways and integratingstimuli into an appropriate transcriptional response at a wide varietyof promoters (3,17). CBP synergizes with SRC-1 in the potentiation ofestrogen receptor and progesterone receptor (PR)-dependenttransactivation (18), indicating a role in nuclear receptor-dependentsignaling. In addition, p300/CBP were among the first regulators ofmammalian transcription in which HAT activity was identified (19).Furthermore, proteins such as the SRC-1 family member p/CIP (8) andthe HAT protein p300/CBP-associated factor (PCAF) (20), firstidentified as binding partners of p300/CBP, have been characterizedas nuclear receptor-associated proteins and coregulators in their ownright (21,22).

The SWI proteins were first identified as potentially importantintermediates in nuclear receptor action when yeast strains bearingmutations inswi genes were found to be incapable ofsupporting glucocorticoid receptor-dependent transactivation (23).Subsequently, human SWI/SNF homologs were found to enhance theactivation functions of glucocorticoid receptor (24) as well asestrogen receptor and retinoic acid receptor (25), and it has beenshown that glucocorticoid receptor directs ligand-dependent nucleosomalremodeling activity of the SWI/SNF complex in yeast (26). Themammalian homologs of the closely related yeastswi2 andsnf2 genes are termedbrahma andbrahma-related gene-1 (brg-1), respectively.BRG-1, the product of thebrg-1 gene, has been shown tointeract with glucocorticoid receptor in a ligand-dependent manner(27), suggesting that mammalian SWI/SNF proteins may be key elementsin nuclear receptor action.

Another subclass of coregulators, relatively undefined functionally,but structurally distinct from those subclasses above, comprises the E3ubiquitin-protein ligases receptor potentiating factor-1 (RPF-1) (28)and E6 papillomavirus protein-associated protein (E6-AP; Z.N.,unpublished work). This subclass of coregulators differs from the SRC-1family and the p300/CBP cointegrators in that they containubiquitin-protein ligase activity rather than HAT activity. They wereinitially identified as factors required for defining substratespecificity in proteolytic degradation by the proteosome system. TheN-terminal receptor activation domains of E6-AP and RPF-1 are separablefrom their ubiquitin ligase domains that reside in their C-terminalHECT. In addition to these characterized subclasses of coregulators, alarge number of receptor-interacting proteins have been identified,including RIP-140 (29), ARA-70 (30), Trip230 (31), and others.

Recently, attention has focused on mechanistic aspects of nuclearreceptor coregulator function, in particular on the nature of thecomplexes that functional evidence indicates they potentially form.Liganded nuclear receptors are reported to recruit a variety ofstructurally diverse proteins: including SRC-1 family members SRC-1(2), GRIP-1/TIF2/SRC-2 (5–7) andp/CIP/RAC3/AIB-1/ACTR/TRAM-1/SRC-3 (8–12); thecointegrators CBP and p300 (3,32); PCAF (21,22); human homologs ofthe yeast SWI/SNF proteins (27) as well as the E3 ubiquitin-proteinligase family members RPF-1 (28) and E6-AP (Z.N., unpublished work). Inaddition, multiple coregulator/coregulator interactions have beenproposed, including p/CIP/CBP (8), CBP/PCAF (20), SRC-1/CBP(3), SRC-1/p300 (33), and SRC-1/PCAF (13). Viewed in theirentirety, these individual observations raise questions concerning thesteady–state organization of coregulators in the cell, as well asaspects of the nature, stability, and molecular relations of theirputative complexes with activated nuclear receptors.

In light of these multiple reported interactions, we decided to addressthe steady–state relationships of multicoregulator transcriptionalcomplexesin vivo by analyzing the biochemical fractionationprofiles of coregulators representative of the different subclassesoutlined above. We demonstrate that different subclasses of nuclearhormone receptor coregulators have distinct fractionation profiles. Wesuggest that at least two members of the SRC-1 coactivator family,SRC-1 and TIF2, can exist in stable complex(es) with each otherin vivo. Furthermore, we provide evidence that PR interactsstably with complexes containing SRC-1 and TIF2 in a ligand-dependentmanner. Our data suggest the existence of discrete, stable subcomplexesof different subclasses of coregulators that may facilitate theassembly of modular complexes required for transcriptional regulationby nuclear receptors.

MATERIALS AND METHODS

Cell Culture and Transient Transfections.

Cell lines wereroutinely maintained at 37°C/5% CO2 in DMEM(HeLa) or RPMI 1640 medium (T47D) supplemented with 5–10%charcoal-stripped fetal calf serum. Transfections were carried outusing Lipofectin (Life Technologies, Gaithersburg, MD). pCR3.1-mCBP wasconstructed by inserting theBamHI-BamHI fragmentof pRcRSV-mCBP8.0 into the corresponding site of pCR3.1 (Invitrogen).The construction of pCR3.1-E6-AP (Z.N., unpublished work),pCR3.1-hSRC-1A, and the reporter pPRE/GRE-E1b-Luc (21) have beendescribed.

Gel Filtration.

Subconfluent T47D or HeLa cells were washedand harvested in PBS and lysed with a disposable manual homogenizer in50 mM NaCl/5 mM KCl/20 mM Hepes, pH 7.5/1 mM EDTA/10% glycerolcontaining a mixture of protease inhibitors (Sigma), and supplementedwith ligand where appropriate. After centrifugation, the supernatantwas loaded on a Superose 6 gel filtration column (Pharmacia)preequilibrated with 150 mM NaCl/50 mM sodium phosphate, pH 7.0(supplemented with ligand where appropriate), and controlled by an FPLCsystem (Pharmacia). For antibody shift experiments, clarified lysateswere rocked for 90 min at 4°C with 1–2 μg of SRC-1 antibody and a3- to 4-fold excess of rabbit anti-mouse IgG (Zymed).

Immunoblotting.

Immunoblotting was performed as described inHansteinet al. (32). Commercially obtained antibodies usedwere anti-CBP (Upstate Biotechnologies, Lake Placid, NY), and anti-RNApolymerase II (RNA pol II) (Santa Cruz Biotechnology).

RESULTS

Subclasses of Nuclear Receptor Coregulators Exist in PrimarilyDistinct Complexesin Vivo.

Our laboratory and othershave previously shown that the functional interaction of nuclearhormone receptors with diverse subclasses of transcriptionalcoactivators is necessary for efficient receptor transactivationin vivo (2–3,5–12,27). Hypothesizing that suchinteractions might require the assembly of multiprotein complexes, weinvestigated the potential existence of nuclear hormone receptorcoactivators in such complexes by biochemical fractionation of T47D andHeLa cell lysates, using a Superose 6 sizing column. Using antibodiesagainst CBP and RNA pol II, we detected endogenous CBP andRNA pol II cofractionating in protein complexes of 1.5–2 MDa (Fig.1), as estimated by Keeetal. (34). The elution profile of RNA pol II was much broader thanthat of CBP (Fig.1; compare fractions 27–30 for CBP with fractions26–34 for RNA pol II), also consistent with previous reports (34). Wethen compared the fractionation profile of endogenous CBPwith that of purified baculovirus-expressed CBP, which elutes as anoligomer in distinct lower molecular size fractions (Fig.1, CBP BACfractions 31–36). This confirmed that CBP in T47D and HeLa cells formshigh molecular weight multiprotein complexesin vivo,consistent with previous reports (34). In addition, the elution patternof p300 in cell lysates closely resembled that of CBP, peaking infractions containing complexes of 1.5–2 MDa, but more detectable inlater fractions than CBP (Fig.2,fractions 27–34).

Figure 1.

Figure 1

High molecular mass complexes contain CBP and RNApol II. Fractionation of T47D lysate on a Superose 6 column wasanalyzed by immunoblot with CBP and RNA Pol II-specific antibodies (CBPand RNA pol II). Recombinant baculovirus-expressed CBP also wasfractionated (CBP BAC). Indicated are elution peaks of molecular massmarkers: mammalian SWI/SNF complex (≈2 MDa) and thyroglobulin (670kDa). The void volume (4 MDa for globular proteins) was determined atfraction 20 by silver staining after fractionation of T47D cell lysate(data not shown).

Figure 2.

Figure 2

Distinct steady–state fractionation profiles ofdifferent subclasses of nuclear receptor coregulators. T47D or HeLacell lysate was fractionated on a Superose 6 column and subjected toimmunoblot analysis by using coregulator-specific antibodies asindicated. Elution peaks of molecular mass standards are indicated. Therelatively sharp elution peaks of SRC-1 and CBP were reproducible. Nodifference in fractionation pattern was observed between different celllines.

We next compared the elution profiles of p300/CBP and RNA pol II withthose of another class of nuclear receptor coregulators, the humanhomologs of the yeast SWI/SNF mediator complex proteins, whichinclude BRG-1, the 220-kDa human homolog of yeast SWI2, and BAF-57, a57 kDa-BRG-1-associatedfactor. Theseproteins exactly cofractionated in complexes of ≥2MDa (Fig.2,peak fractions 25–27), consistent with previous estimates (35,36). Adistinct, second peak of BAF-57 was observed in later fractions (Fig.2, peak fraction 38). Longer exposures of the BRG-1 immunoblots (datanot shown) indicated that minor amounts of BRG-1 copurified with thepeak fractions of SRC-1 and TIF2 (see below).

In the light of previous reports of an interaction between thecointegrators p300/CBP and PCAF (20), we next examined whether PCAFpeaked in the same fractions in which p300/CBP peaked. PCAF was foundto peak slightly earlier than the elution peaks of p300/CBP (Fig.2,peak fractions 27–28), indicating that PCAF is not an exclusivebinding partner of p300 and may form complexes with other proteins suchas the human homologs of the SWI/SNF complex. A second minor pool ofPCAF was observed (Fig.2, fractions 35–37), which may representpartially dissociated PCAF complexes, or complexes with factors otherthan p300/CBP. These PCAF pools were variable in proportion betweenruns (data not shown), and exhibited the greatest variation of allcoregulators analyzed.

Because several studies have suggested that SRC-1 may exist incomplexes with CBP (3) (Fig.2, elution peak fractions 29–30), p300(33) (Fig.2, elution peak fractions 28–30) and PCAF (13) (Fig.2,major elution peak fractions 27–28), we next analyzed the elutionprofile of SRC-1 in relation to these proteins. Analysis of thefractionation pattern of SRC-1 showed that it peaked sharply infractions containing protein complexes of an estimated 0.5–0.6 MDa(Fig.2, fractions 33–35). Overlap between the elution patterns ofSRC-1 and CBP was undetectable (Fig.2), implying that these proteinsmay exist in distinct preformed complexes, contrary to previous reports(3). In contrast, the elution pattern of SRC-1 overlapped slightly withminor pools of p300 and PCAF (Fig.2), suggesting that should stablecomplexes between SRC-1 and these coregulators exist, they representonly a small proportion of their respective cellular pools.

Monomeric SRC-1 was undetectable in cell lysates, suggesting that thekinetics of the complex formation strongly favor the sequestration ofSRC-1 in these complexes, or that the free form is subject to rapiddegradation. As a control, we fractionated baculovirus-expressed SRC-1by Superose 6 gel filtration and found that it eluted exclusively infractions 32–35 (data not shown), similar to its elution profile incell lysate (Fig.2, lanes 33–35) that might indicatehomomultimerization of SRC-1, but also may be attributable toincomplete purification of recombinant SRC-1 from insect cellcoregulators. Similar to its elution profile in T47D and HeLa celllysate, no monomeric purified SRC-1 was detectable, further suggestingthat the free form of SRC-1 may be kinetically unstable. We thenexamined the elution profile of a second member of the SRC-1 family,TIF2. TIF2 copurified with SRC-1, although its elution pattern was lessdefined and covered a wider range of fractions than SRC-1 (Fig.2,fractions 31–36). No cross reactivity was observed between the SRC-1antibody and TIF2 in immunoblots (not shown). The relatively broadelution profile of TIF2 suggests that it might form a greater varietyof complexes than its family member SRC-1.

These initial observations suggested to us that different subclasses ofcoactivator involved in nuclear receptor transactivation might besequestered in largely distinct complexes. To further test thishypothesis, we examined the elution profiles of two members of a lesswell-defined but functionally distinct subclass of nuclear hormonecoregulators, the E3 ubiquitin-protein ligases RPF-1 and E6-AP. E6-APand RPF-1 proteins were observed to copurify in complexes of 200–300kDa and are distinct from all of the complexes previously observed(Fig.2, fractions 38–41).

E6-AP and RPF-1 Synergistically Enhance PR Transactivation.

The copurification of E6-AP and RPF-1 by Superose gel filtrationsuggested to us that they might be present in common complexes. To testtheir possible functional interaction, we next examined whether thesecoactivators might synergistically enhance transactivation by PR. HeLacells were transiently cotransfected with E6-AP/RPF-1, E6-AP/SRC-1,and E6-AP/CBP in a luciferase-based PR reporter assay (Fig.3). Whereas the combinations ofE6-AP/CBP (Fig.3a) and E6-AP/SRC-1 (Fig.3b)only additively enhanced PR transactivation, E6-AP and RPF-1 (Fig.3c) synergistically enhanced PR transactivation.

Figure 3.

Figure 3

Synergistic enhancement of PR transactivation byE6-AP and RPF-1. HeLa cells were transiently transfected with 0.2 μgof PR-B expression plasmid and 1 μg of pPRE-E1b-Luc reporter in thepresence and absence of 0.5 μg (total) of vectors expressing theindicated coactivators. The cells were treated with either vehicle only(−R5020) or 10nM R5020 (+). Data are expressed as the mean (± SD) oftriplicate values.

Association of SRC-1 and TIF2 in a Single ComplexinVivo.

While the copurification of SRC-1 and TIF2 wasevidence that they might form a complexin vivo (Fig.2), weverified this by incubating cell lysate with anti-SRC-1 antibody andrabbit anti-mouse IgG before fractionation on the Superose 6 column. Asanticipated, this resulted in a clear shift of SRC-1 immunoreactivityto fractions containing significantly larger protein complexes (compareFig.4 without anti SRC-1antibody, fractions 33–35 and Fig.4 with anti-SRC-1 antibody,fractions 28–32). Stripping and reprobing the same blot with anti-TIF2antibody indicated a considerable shift of the immunoreactive TIF2 intofractions containing shifted SRC-1 (compare Fig.4 without SRC-1antibody, fractions 31–36 and Fig.4 with anti-SRC-1 antibody,fractions 28–32). To demonstrate that the coeluting of TIF2 andshifted SRC-1 was not due to nonspecific primary or secondary antibodybinding, the blot was stripped and reprobed with anti-CBP antibodydemonstrating that CBP eluted in the same fractions irrespective ofpreincubation of lysate with SRC-1 antibody (data not shown). Becausethe monoclonal SRC-1 antibody does not cross react with TIF2, we takethese results to indicate that TIF2 and SRC-1 can form commoncomplexes. As shown earlier, the broader fractionation profile of TIF2with respect to SRC-1 (Fig.2) indicates that TIF2 likely also existsin complexes distinct from that which it forms with SRC-1. This issupported by the fact that, although incubation with SRC-1 antibodyresults in significant shift in the SRC-1 elution profile, a proportionof TIF2 is not shifted by anti-SRC-1 antibody (Fig.4). Taken together,our results indicate that SRC-1 family members may associate with eachother in heteromultimeric protein complexes.

Figure 4.

Figure 4

SRC-1 and TIF2 can form common complexesin vivo. SRC-1 complexes were collected by incubationwith SRC-1 monoclonal antibody and polyclonal antimouse IgG andfractionated by gel filtration. Immunoblotting confirmed the shift ofSRC-1 from its elution peak in the absence of preincubation withanti-SRC-1 antibody (−) to earlier fractions in the presence ofanti-SRC-1 antibody (+). The relatively broad elution profile ofshifted SRC-1 is most likely due to the heterogeneity of immunecomplexes formed in these fractions.

Liganded PR Recruits Preformed Complexes Containing SRC-1 and TIF2in Vivo. To address the relationship of nuclearreceptor with these coregulator complexes, we examined their relativemigration patterns in the presence and absence of ligand.

T47D cellswere used for these experiments given their elevatedendogenous levels of PR. Lysate from cells pretreated withvehicle or with hormone was subjected to fractionation on the Superosecolumn. Unliganded PR A and B forms eluted in fractions containingprotein complexes in the range of ≈500-kDa (Fig.5a,i,lanes 32–39, longer exposure of 5a,i, lanes32–41), masses consistent with previous reports (1,37). In thepresence of hormone, the liganded PR-B form copurified sharply with theelution peaks of SRC-1 and TIF2 (Fig.5a,ii,lane 34; compare with Fig.2, SRC-1 and TIF2). The liganded PR A formalso coeluted with the peaks of SRC-1 and TIF2 but significant amountsdid not (Fig.5a,ii, lanes 36–41). Liganded PRwas largely absent from fractions in which the majority of cellularp300/CBP eluted (compare Fig.5a,ii with Fig.2, p300/CBP).

Figure 5.

Figure 5

Liganded PR exists in stable complexes containingSRC-1 and TIF2in vivo. (a) T47D cellswere pretreated with vehicle(i) and with 1nMprogesterone (ii) before fractionation andimmunoblotting with PR antibody. (b) Cells were treatedas above except lysate was incubated with anti-SRC-1 antibody,fractionated and immunoblotted for (i) PR,(ii) SRC-1, (iii) PR, (iv)SRC-1, and (v) TIF2. (The arrow indicates the peak ofSRC-1 and TIF2 in the absence of preincubation with the SRC-1antibody).

The presence of the liganded PR forms in fractions containing the peaksof SRC-1 and TIF2 was not conclusive evidenceper se of anassociation of PR, SRC-1, and TIF2. To address more precisely theassociation of liganded PR with the SRC-1 and TIF2-containing complexesin vivo, we incubated SRC-1 antibody and polyclonalanti-mouse IgG with T47D lysates prepared from cells pretreated withand without hormone. After fractionation of T47D lysate preincubatedwith SRC-1 antibody, the elution pattern of the unliganded PR forms waslargely unaltered (compare Fig.5b,i with Fig.5a,i), but SRC-1 was shifted to earlierfractions as predicted (Fig.5b,ii, lanes29–32). In contrast, after ligand treatment of T47D cells,preincubation of lysate with SRC-1 antibody resulted in the shifting of60–70% of liganded PR A and B forms (Fig.5b,iii, lanes 28–31) into fractions containing supershiftedSRC-1 (Fig.5b,iv, lanes 28–31) and TIF2 (Fig.5b,v, lanes 30–31). The relatively broadelution profile of shifted liganded PR (compare Fig.5b,iii with Fig.5a,ii) is most likelydue to the heterogeneity of immune complexes formed in these fractions.A significant proportion of liganded PR A and B forms was not shifted(Fig.5b,iii), suggesting that liganded PR alsomay exist in complexes that do not bind SRC-1 antibody. Our datasuggest that,in vivo, complexes containing SRC-1 and TIF2associate stably with PR A and B forms in a ligand-dependent manner.

DISCUSSION

The formation of coregulatorsomes, or multicoregulator complexes,at hormone-regulated promoters has been widely postulated on the basisof multiple interactions between nuclear receptors and coregulators.Inferences as to the nature of the associations within these complexeshave been founded largely on functional assays. In particular, thequestion has been raised of whether these coregulatorsomes associate inthe steady–state or whether pools of specific precursor complexesexist. Our data provide direct evidence of the existenceinvivo of stable subcomplexes of distinct nuclear receptorcoregulator subclasses, possibly reflecting established functionaldifferences between these subclasses of coregulators. We suggest thatthis physical partition of different subclasses of coactivators affordsthe potential for their efficient combinatorial assembly into higherorder complexes. This is consistent with the functional data of Korzuset al. (38), which suggest that the requirement for maximaltranscriptional activation at specific promoters may be a function ofthe existence of diverse groups of coactivator complexes. From ourdata, it is plausible that transient interactions between the stablesubcomplexes we have observed would facilitate rearrangement of finalcoregulator complexes into multiple configurations.

One issue that is unclear from our data is whether the complexes wehave observed represent component parts of larger transcriptionalcomplexes, the kinetics of formation of which do not withstand ourexperimental conditions. Coimmunoprecipitation andin vitroexperiments have detected interactions between SRC-1 and othersubclasses of nuclear receptor coregulators such as p300 (32), CBP (3),and PCAF (13), as well as interactions between receptor and CBP (3),p300 (32), BRG-1(27), and PCAF (13). Our assay differs from theseexperiments in that we have been able to analyze multiple coregulatorcomplexes in terms of the relative strengths of their steady–stateinteractions. In our assay, while SRC-1 was undetectable in fractionscontaining CBP (Fig.2), we did observe some overlap of SRC-1 withminor pools of p300 and PCAF (Fig.2). Interestingly, we also were ableto copurify SRC-1 and small amounts of BRG-1 (Fig.2), raising thepossibility that these coregulators form stable steady–statecomplexes. Our data indicate however that putative complexes betweenSRC-1/BRG-1, SRC-1/p300, PR/BRG-1, and SRC-1/PCAF, in thesteady–state of the cell, represent only small pools of the totalamount of these proteins in the cell. In the context of our assay, itis possible that “final” transcriptional complexes are disruptedinto the smaller, stable subcomplexes we have observed. However, wehave reproduced the elution pattern of previously established complexesunder our experimental conditions, such as the mammalian SWI/SNFcomplex (35,36). Because we do not observe them under our conditions,final complexes comprised of different subcomplexes may be inherentlylabile and subject to rapid rearrangement, a plausible mechanism offine control at transcriptionally active promoters. Additionally, wehave not yet detected monomeric forms of coregulatorsinvivo, suggesting that an important mechanism of control oftranscription may be the kinetic instability of the monomeric forms ofcoregulators.

The identification of the stable association of SRC-1 and TIF2 in asingle complex, as well as the ability of SRC-1 to homomultimerize,suggests that protein-protein interactions between SRC-1 family membersis important for their functionin vivo. The sequenceconservation between family members within the Per-Arnt-Sim/basichelix–loop–helix homology domains, taken together with our data,lends credence to the possibility that the Per-Arnt-Sim/basichelix–loop–helix homology domains mediate this interaction, but thisis yet to be established. One consequence of this multimerization mightbe to increase the number of binding interfaces at which afferentsignaling pathways might integrate with promoter-bound receptor.

The precise copurification of the functionally related coactivatorsE6-AP and RPF-1 in 200–300 kDa complexes is evidence that theseproteins may form a stable complexin vivo. In light of thecooperative enhancement of PR transactivation by E6-AP and RPF-1, butnot E6-AP/SRC-1 and E6-AP/CBP, we speculate that the putativephysical association of E6-AP and RPF-1 in common complexes may berelated to their synergism. Interestingly, SRC-1 and TIF2, while theycan form common complexes, do not synergistically enhancetransactivation by PR (data not shown). We suggest this anomaly is dueto the fact that E6-AP and RPF-1 have different downstream targets,E6-AP being involved in p53 and HHR23A ubiquitination (39,40), whereasRPF-1 is required for RNA pol II ubiquitination (41). Conversely, theHAT activities of SRC-1 and TIF2 probably have similar downstreamchromatin targets and are likely to be redundant in cotransfectionassays. Further studies are required to establish more clearly whetherthe mechanistic basis of the synergism of E6-AP and RPF-1 is related totheir possible existence in a common complex.

Our demonstration of the ligand-dependent association of PR with theSRC-1/TIF2 complex is the first direct evidence that liganded PRassociates stably with large coregulator complexes as a distinct stepin transactivationin vivo. We have shown that unliganded PRforms stable complexes over the range of 400–500 kDa, consistent,within the error of the column, with previous estimates for unligandedPR complexes (1,37). Liganded PR associates stably with similar sizedcomplex(es) that contain SRC-1 and TIF2. The interaction betweenactivated PR and SRC-1/TIF2 complexes that we have demonstrated isclearly a stable interactionin vivo, in comparison to anyinteraction with CBP or p300. Because liganded PR did not coelute withthe major elution peaks of CBP or p300 in the context of our assay, wesuggest that activated PR does not recruit these proteins in a stablecomplex. Rather, our data indicate that liganded PR associates stablywith the major peaks of SRC-1 and TIF2, indicating that the complexeswithin these fractions may represent important fundamentalintermediates in PR transactivation. Although our assay is not open tofunctional interpretation, it is possible that these stablePR/SRC-1/TIF2 complexes undergo relatively transient interactionswith other subclasses of coregulators during transcriptionalregulation. Our laboratory has suggested (42) that subsequent toformation of a stable committed complex, a “rapid-start” complexis assembled by liganded PR for subsequent rounds of transcription of atemplate. The relative stability of the liganded PR/SRC-1/TIF2complexes, makes them plausible candidates for such a rapid-startcomplex. To further support such a notion, it has been shown that thefunctional requirement of p300 for estrogen receptor transactivationin vitro is reduced before transcriptional reinitiation(43), suggesting that the interaction of p300 with liganded estrogenreceptor may be relatively transient. Future work will clarify thefunctional components of the complexes with which activated PRassociates stablyin vivo.

Transcriptional regulation by nuclear receptors is increasingly beingseen as a modular process, involving multiple discrete steps, such aschromatin remodeling and recruitment of basal transcription factors(27,43,44). As a mechanistic basis for this, the multiple distinctsubcomplexes we have identified here afford the possibility for theirstepwise, sequential interactions with liganded receptor duringtranscriptional activation. A model based on our data (Fig.6) suggests that hierarchicalinteractions, of varying stability, may contribute to transcriptionalregulation by PR and coregulators. In our model, liganded PR, SRC-1,and TIF2 are present in comparatively stable core complexes thatundergo relatively transient associations with other subcomplexesduring transcriptional initiation. In support of such a notion, Fondellet al. (45) have identified a class of thyroidreceptor-interacting proteins that copurify with constitutivelyliganded thyroid receptor. These thyroid receptor-interacting proteinsare distinct from any coregulator class previously characterized andindicate that liganded receptor may undergo sequential interactionswith different multiprotein complexes during transcriptionalregulation. Future work will discern the functional significance ofthese and other complexes and their roles in regulation of geneexpression by nuclear receptors.

Figure 6.

Figure 6

Mechanistic model for transcriptional activationby activated PR. The relative stability of the complexes betweenliganded PR and SRC-1/TIF2-containing subcomplexes suggests they maybe important intermediates in PR transactivation. Interactions of SRC-1with other subclasses of coregulators appear to be comparativelytransient.

Acknowledgments

We thank members of the O’Malley laboratory for helpfuldiscussions and critical reading, M. Burcin and J. Wong for recombinantproteins, D. Edwards for the E6-AP and SRC-1 antibodies, N. Weigel forthe PR antibody, D. McDonnell for the RPF-1 antibody and RPF-1construct, P. Chambon for the TIF2 antibody, R. Eckner for the p300antibody, W. Wang for the BRG-1 and BAF-57 antibodies, Y. Nakatani forthe PCAF antibody, and R. Goodman for the CBP construct. This work wassupported by a National Institutes of Health grant.

ABBREVIATIONS

SRC-1

steroid receptor coactivator-1

CBP

CREB-binding protein

BRG-1

product of thebrg-1 gene

E6-AP

E6 papillomavirus protein-associated protein

RPF-1

receptor-potentiating factor-1

TIF2

transcription intermediary factor2

PCAF

p300/CBP-associated factor

RNA pol II

RNA polymerase II

PR

progesterone receptor

HAT

histone acetytransferase

References

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